Bioprosthetic heart valves (BHVs) derived from glutaraldehyde crosslinked porcine aortic valves are used in thousands of heart valve replacement surgeries. These devices often fail clinically due degeneration and pathologic calcification. In recent years others and we have shown that valvular glycosaminoglycans (GAGs) present in the middle spongiosa layer are lost during tissue fixation and after implantation. Maintaining the structural integrity of the extracellular matrix (ECM) in those processed tissues is the quintessence of a durable BHV. The overall aim of this project is to better understand the role of GAGs in the pathology of BHVs and to increase their retention and stabilization in the valvular ECM. The long-term goal of our research is to improve BHVs function by increasing the retention and stabilization of valvular GAGs so that these devices would function for extended time periods. We hypothesize that the loss of GAGs from the extracellular matrix of glutaraldehyde crosslinked BHVs causes a reduction in bending stiffness and collagen structural deterioration. We further hypothesize that proper GAG stabilization strategies would better preserve the heart valve structure and improve the mechanical properties of the valve, leading to less degeneration during its function. Moreover, based on the GAGs role in physiologic calcification and our preliminary studies, we hypothesize the preservation of these molecules will aid in the prevention of cuspal calcification. We will pursue following aims 1) We will monitor the status of valvular GAGs during tissue harvesting, preparation and glutaraldehyde (GA) fixation and after implantation in an animal model. We will also study the mechanisms by which tissue GAGs are lost from BHVs, focusing on GAG-degrading enzymes as potential candidates. 2) We will chemically manipulate the tissues during the critical stages of preparation to improve retention and stabilization of valvular PGs. We will optimize our novel periodate-based crosslinking procedure for maximum retention and stabilization of valvular GAGs by determining the most advantageous reaction parameters. The crosslinking efficacy of the periodate procedure will be enhanced by including agents such as diamines. 3) We will investigate the mechanical properties of tissues in which GAGs were stabilized with the optimized periodate procedure. Mechanical stability of BHV ECM including GAGs will be monitored by undertaking in vitro cyclic fatigue. Furthermore, we will evaluate the biostability of GAGs and resistance to calcification of BHVs of optimally stabilized valvular tissues by in vivo rat subdermal implantation model.